Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility

Abstract

Small multidrug resistance (SMR) transporters perform coupled antiport of protons and toxic substrates, contributing to antibiotic resistance through efflux of these compounds from the bacterial cytoplasm. Extensive biophysical studies of the molecular transport mechanism of the E. coli SMR transporter EmrE indicate that it should also be capable of performing proton/drug symport or uniport, either of which will lead to drug susceptibility rather than drug resistance in vivo. Here we show that EmrE does indeed confer susceptibility to some small molecule substrates in the native E. coli in addition to conferring resistance to known polyaromatic cation substrates. In vitro experiments show that substrate binding at a secondary site triggers uncoupled proton uniport that leads to susceptibility. These results suggest that the SMR transporters provide one avenue for bacterial-selective dissipation of the proton-motive force. This has potential for antibiotic development and disruption of antibiotic resistance due to drug efflux more broadly.

Functional promiscuity of small multidrug resistance transporters fromStaphylococcus aureus,Pseudomonas aeruginosa, andFrancisella tularensis

“…These results show some variation in specific substrate profile for each SMR homolog, as expected based on sequence variation in regions of the transporter known to be important for substrate binding and specificity. Although the results of the growth and metabolic assays characterizing the ability of SMR transporters to confer susceptibility or resistance to different compound in E. coli are not perfectly consistent, these orthogonal assays confirm that the functional promiscuity previously described for EmrE (34) does extend to at least three other SMR homologs across the same subfamily.…”

“…Many known SMR substrates are polyaromatic cations, and this commercially available assay is designed to avoid interference from the natural fluorescence of such compounds, enabling a single consistent assay format for characterization across several hundred potential substrates selected without bias towards common MDR efflux pump substrates. Our recent Biolog phenotypic microarray screen of E. coli revealed that EmrE confers resistance or susceptibility depending on the identity of the substrate, an unprecedented result (34). Here we repeat this screen on SAsmr, PAsmr, and FTsmr expressed in MG1655 Δ emrE E. coli cells.…”

“…We previously demonstrated that EmrE confers susceptibility to harmane because harmane binds to the transporter and triggers uncoupled proton flux (34). In other words, EmrE acts as a harmane-gated proton uniporter, but it functions as a proton-coupled antiporter of ethidium and other resistance substrates.…”

“…The original mechanistic model of coupled 2 H + : 1 toxin antiport, was based on early experimental data showing electrogenic transport of a +1 toxin and electroneutral transport of a +2 toxin (31). More recent mechanistic studies have demonstrated that EmrE is not a strictly coupled proton/drug antiporter in the manner of the GdX-subfamily and may be able to perform uniport and symport (32, 34) in addition to antiport. This new free exchange model of EmrE transport has major biological implications, since any of the alternative transport modes should lead to enhanced susceptibility , rather than resistance to the small molecule substrate (34).…”

Still rocking in the structural era: A molecular overview of the small multidrug resistance (SMR) transporter family

“…Moreover, structural homology with SLC35/DMT transporters suggests that, contrary to the proposal that the SMRs are unusual in having not evolved fused, inverted repeat architecture ( 29 ), it is probable that the SMR fold has indeed been preserved through this evolutionary mechanism, albeit with a helical insertion and domain swap along the way. These recent advances in understanding the molecular architecture bolster ongoing efforts to develop antimicrobials that target SMR proteins, either by inhibiting transporter assembly in order to sensitize bacteria to transported compounds ( 78 , 137 , 138 ) or by hijacking the nominal exporters to import antimicrobial compounds instead ( 139 ).…”